Catalytic antibodies and a method of producing same

ABSTRACT

The present invention relates generally to a growth factor precursor and its use to select production of antigen specific catalytic antibodies. Such catalytic antibodies are produced following B cell activation and proliferation induced by catalytic cleavage products of a target antigen portion of the growth factor precursor of the present invention. A particularly useful form of the growth factor precursor is as a nucleic acid vaccine. The nucleic acid vaccine of the present invention preferably further comprises a molecular adjuvant. Another aspect of the present invention comprises a growth factor precursor in multimeric form. The growth factor precursor of the present invention is useful for generating catalytic antibodies for both therapeutic, diagnostic and industrial purposes.

FIELD OF THE INVENTION

The present invention relates generally to a growth factor precursor andits use to select production of antigen specific catalytic antibodies.Such catalytic antibodies are produced following B cell activation andproliferation induced by catalytic cleavage products of a target antigenportion of the growth factor precursor of the present invention. Aparticularly useful form of the growth factor precursor is as a nucleicacid vaccine. The nucleic acid vaccine of the present inventionpreferably further comprises a molecular adjuvant. Another aspect of thepresent invention comprises a growth factor precursor in multimericform. The growth factor precursor of the present invention is useful forgenerating catalytic antibodies for both therapeutic, diagnostic andindustrial purposes.

BACKGROUND OF THE INVENTION

The rapidly increasing sophistication of recombinant DNA technology isgreatly facilitating research and development in the medical and alliedhealth fields. A particularly important area of research is the use ofrecombinant antigens to stimulate immune response mechanisms andoutcomes. However, recombinant techniques have not been fully effectivein generating all components of the humoral response. One such importantyet not fully exploited component is the catalytic antibody.

Catalytic antibodies are highly substrate specific catalysts which canbe used, for example, to proteolytically activate or inactivateproteins. Catalytic antibodies have great potential as therapeuticagents in human diseases such as rheumatoid arthritis, AIDS andAlzheimer's disease amongst many others.

Antibody therapy has been used in patients Antibodies have a half-lifeof about 23 days in the circulation of humans which is a clear advantageover other drugs. Catalytic antibodies, however, are considered to beeven more effective. They are recycled after their antigenic encounterand are not bound to the antigen as occurs with “classical” antibodies.Catalytic antibodies should, therefore, function at a much lower dosethan classical antibodies and could be used at sub-immunogenic doses.Catalytic antibodies would be particularly useful in long term therapy.

Traditionally, catalytic antibodies have been generated by immunisingmice with transition state analogs. Such antibodies have been shown tocatalyse several chemical reactions. However, this approach has a severelimitation in that it is difficult to predict the structure oftransition state analogs which effect proteolysis of specific proteins.Immunising a mouse with a transition state analog is by definitioninefficient since it selects B cells on the ability of surfaceimmunoglobulins to bind the analogs and not on the ability of a surfaceimmunoglobulins to catalytically cleave the analogue. This is one of thereasons why catalytic antibodies have relatively low turn-over rates andcannot compete with the naturally occurring enzyme counterparts, in thecase where they exist.

Another approach has been the mutation of conventional antibodies toalter their activity to be catalytical like in nature. However, to date,such an approach has not proved successful.

As a consequence, catalytic antibodies have not previously achievedprominence as therapeutic, diagnostic or industrial tools.

There is a need, therefore, to develop a more efficacious approach togenerating catalytic antibodies having desired catalytic specificity.

International Patent Application No. PCT/AU97/00194 filed on 26 Mar.1997 and is herein incorporated by reference provided a means forselecting catalytic B cells. The method contemplated a growth factorcomprising two Ig binding domains from protein L of Peptostreptococcusmagnus as B cell surface molecule binding portions flanking a T cellsurface molecule binding portion (designated “H”) from hen egg lysozyme(HEL). The specificity of the LHL growth factor for catalytic B cellswas provided by an antigen masking or attached to a molecule masking oneor more of the B cell surface molecule binding portions. Catalyticcleavage of the antigen exposed the B cell surface molecule bindingportions to permit catalytic antibody production.

In accordance with the present invention, there is provided an improvedgrowth factor precursor.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Sequence Identity Numbers (SEQ ID NOs.) for nucleotide and amino acidsequences referred to herein are defined following the Abbreviations.

One aspect of the present invention is directed to a growth factorprecursor comprising a recombinant polypeptide chain or a moleculehaving modular peptide components or a synthetic equivalent thereofwherein said polypeptide chain or modular peptide molecule comprises atleast one B cell surface molecule binding portion, at least one T cellsurface molecule binding portion capable of providing T cell dependenthelp to a B cell, an antigen cleavable by a catalytic antibody and apeptide portion comprising domains from both a variable heavy chain anda variable light chain of an immunoglobulin and wherein said variableheavy chain and variable light chain domains in the growth factorprecursor, associate together by intra- and/or inter-domain bonding andsubstantially prevent the at least one B cell surface molecule bindingportion from interacting with a B cell surface molecule such that uponcleavage of said antigen by a catalytic antibody, the peptide comprisingsaid variable heavy chain and variable light chain domain permits the atleast one B cell surface molecule binding portion to interact with a Bcell surface molecule.

Another aspect of the present provides a growth factor precursorcomprising a recombinant polypeptide chain or a molecule having modularpeptide components or a synthetic equivalent thereof wherein saidpolypeptide chain or modular peptide molecule comprises at least one Bcell surface molecule binding portion, at least one T cell surfacemolecule binding portion capable of providing T cell dependent help to aB cell, an antigen cleavable by a catalytic antibody and a peptideportion comprising domains from both a variable heavy chain and avariable light chain of an immunoglobulin and wherein said variableheavy chain and variable light chain domains in the growth factorprecursor associate together by intra- and/or inter-domain bonding andsubstantially prevent the at least one B cell surface molecule bindingportion from interacting with a B cell surface molecule such that uponcleavage of said antigen by a catalytic antibody, the peptide comprisingsaid variable heavy chain and variable light chain domain permits the atleast one B cell surface molecule binding portion to interact with a Bcell surface molecule wherein if said growth factor precursor comprisesa single B cell surface molecule binding portion, then the growth factorprecursor further comprises a multimerising inducing element.

Yet another aspect of the present invention provides a growth factorprecursor comprising a recombinant polypeptide chain or a moleculehaving modular peptide components or a synthetic equivalent thereofwherein said polypeptide chain or modular peptide molecule comprises atleast two B cell surface molecule binding portions, at least one T cellsurface molecule binding portion capable of providing T cell dependenthelp to a B cell, an antigen cleavable by a catalytic antibody and apeptide portion comprising domains from both a variable heavy chain anda variable light chain such that in the growth factor precursor, thesevariable chain domains associate together by intra- and/or inter-domainbonding and, when associated together, substantially prevent at leastone of the B cell surface molecule binding portions from interactingwith a B cell surface molecule wherein upon cleavage of said antigen bya catalytic antibody, the at least two B cell surface molecule bindingportions induce activation and proliferation of a B cell expressing saidcatalytic antibody.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation showing the structure of LgLcomprising ompA and the hexa-his-Tag on the C terminus.

FIG. 2 is a photographic representation showing production of OHLgL inE. coli using 20% w/v PHAST-gels.

FIG. 3 is a graphical representation, of the 280 nm absorbance traceshowing purification of LgL on a HPLC superose 12 column.

FIG. 4 is a photographic representation of LgL fractions from a HPLCsuperose 12 column on a 20% w/v PHAST gel.

FIG. 5 is a graphical representation showing biological potency of LgLas demonstrated by B7-1 and B7-2 expression after overnight stimulation.

FIG. 6 is a diagrammatic representation showing structure of ccMTLgLcomprising LgL with TEV cleavage signal and disulphide linked singlechain Fv from McPc603.

FIG. 7 is a photographic representation of ccMTLgL containing fractionsfrom a FLAG M1 affinity column analysed on a PHAST-gel.

FIG. 8 is a graphical representation of the 280 nm absorbance trace offractions containing ccMTLgL from an HPLC superose 12 gel.

FIG. 9 is a photographic representation of ccMTLgL fractions from HPLCsuperose 12 gel analysed on PHAST gel.

FIG. 10 is a photographic representation showing presence ofinter-domain disulphide bond in ccMTLgL on 20% w/v PHAST gel underreducing and non-reducing conditions, before and after cleavage withTEV.

FIG. 11 is a graphical representation showing B7-1 expression afterovernight stimulation of mesenteric lymph node cells with anti-μ, LgL,ccMTLgL and ccMTLgL+TEV.

FIG. 12 is a graphical representation showing the results of repeatingthe experiment associated with FIG. 11 except that TEV is also added insitu to the overnight B cell cultures.

FIG. 13 is a schematic representation of ompL.

FIG. 14 is a schematic representation of Fv-catAb.

FIG. 15 is a photographic representation of a silver stained 20% w/vPAGE SDS PHAST-gel analysis of scM603 purified from periplasmic fractionvia an L-column.

The following abbreviations are used in the specification.

-   ccMTLgL Growth factor precursor comprising LgL linked to variable    heavy and light chain domains from antibody McPc603 via TEV    sensitive peptide-   FSC Forward light scatter-   g Glycine-serine linker having the structure (GGGGS)₄-   H T cell surface molecule binding portion from hen egg lysosyme    (HEL)-   hulgG Human immunoglobulin G-   L B cell surface molecule binding portion from protein L of    Peptostreptococcus magnus-   LgL Two L molecules linked via glycine-serine peptide-   LHL Growth factor comprising H flanked by two L molecules-   McPc603 Antibody having anti-phosophorylcholine specificity-   TLHL LHL linked to kappa light chain via TEV sensitive peptide and g    attached to N terminus region

SUMMARY OF SEQ ID NOs.

SEQ ID NO. MOLECULE Nucleotide Amino acid LHL 1 2 CATAB-TEV 3 4 TLHL 5 6LHL.seq 7 8 FLAG epitope — 9 Kappa 10 11 LHL-omp 12 13 Strep-tag — 14ccMTLgL 15 16

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides in part an improved growth factorprecursor capable of selecting catalytic B cells. The selected catalyticB cells then undergo mitogenesis including activation and proliferationas a pre-requisite for the production of catalytic antibodies.

Accordingly, one aspect of the present invention is directed to a growthfactor precursor comprising a recombinant polypeptide chain or amolecule having modular peptide components or a synthetic equivalentthereof wherein said polypeptide chain or modular peptide moleculecomprises at least one B cell surface molecule binding portion, at leastone T cell surface molecule binding portion capable of providing T celldependent help to a B cell, an antigen cleavable by a catalytic antibodyand a peptide portion comprising domains from both a variable heavychain and a variable light chain of an immunoglobulin and wherein saidvariable heavy chain and variable light chain domains in the growthfactor precursor associate together by intra- and/or inter-domainbonding and, when associated together, substantially prevent the atleast one B cell surface molecule binding portion from interacting witha B cell surface molecule such that upon cleavage of said antigen by acatalytic antibody, the peptide comprising said variable heavy chain andvariable light chain domain permits the at least one B cell surfacemolecule binding portion to interact with a B cell surface moleculewherein if said growth factor precursor comprises a single B cellsurface molecule binding portion, then the growth factor precursorfurther comprises a multimerising inducing element.

The present invention further provides a growth factor precursorcomprising a recombinant polypeptide chain or a molecule having modularpeptide components or a synthetic equivalent thereof wherein saidpolypeptide chain or modular peptide molecule comprises at least one Bcell surface molecule binding portion, at least one T cell surfacemolecule binding portion capable of providing T cell dependent help to aB cell, an antigen cleavable by a catalytic antibody and a peptideportion comprising domains from both a variable heavy chain and avariable light chain of an immunoglobulin and wherein said variableheavy chain and variable light chain domains in the growth factorprecursor associate together by intra- and/or inter-domain bonding and,when associated together, substantially prevent the at least one B cellsurface molecule binding portion from interacting with a B cell surfacemolecule such that upon cleavage of said antigen by a catalyticantibody, the peptide comprising said variable heavy chain and variablelight chain domain permits the at least one B cell surface moleculebinding portion to interact with a B cell surface molecule wherein ifsaid growth factor precursor comprises a single B cell surface moleculebinding portion, then the growth factor precursor further comprises amultimerising inducing element.

The growth factor precursor is deemed a “precursor” since it issubstantially incapable of inducing B cell mitogenesis (i.e. activationand proliferation followed by antibody production) in the absence ofcatalytic cleavage of a portion of the growth factor precursor whichmasks at least one B cell surface molecule binding portion on themolecule. By masking the B cell surface molecule binding portion, thegrowth factor precursor is substantially incapable of inducing B cellmitogenesis such as by, but not limited to, cross-linking of B cellsurface immunoglobulins. The term “masks” or “masking” includes thesteric, conformational, electrostatic and/or physical interference at orproximal to at least one B cell surface molecule binding portion on thegrowth factor precursor thus preventing interaction between the B cellsurface molecule binding portion and a B cell surface molecule. One ofthe catalytic products of the growth factor precursor of the presentinvention is a growth factor capable of inducing B cell mitogenesis.

The growth factor precursor of the present invention may be synthesisedas a single polypeptide chain. The polypeptide chain comprises variousregions such as a component of the variable heavy chain and a componentof a variable light chain of an immunoglobulin (referred to herein asvariable light chain and variable heavy chain domains), a targetantigen, a T cell surface molecule binding portion and at least one Bcell surface molecule binding portion. Additional regions may also beincluded such as purification tags including FLAG and hexa-his and amolecular adjuvant such as but not limited to C3d, CTLA4 and/or CD40L.Such a polypeptide may be produced from fusing together a series ofnucleotide sequences to produce a single nucleic acid molecule which,when expressed in an appropriate host cell, produces a single amino acidsequence in the form of the polypeptide.

Alternatively, the polypeptide chain may be made in modular form and themodules bound, ligated, linked or otherwise associated together. Forexample, the growth factor precursor may comprise a multimodularmolecule having a module comprising a B cell surface molecule bindingportion, a module comprising a T cell surface molecule binding portion,and one or more modules comprising the variable heavy chain domain andvariable light chain domain.

The modular components may be bound, ligated or otherwise associatedtogether by any convenient means such as but not limited to peptidebonding, electrostatic attraction, covalent bonding, di-sulphide bridgesand/or hydrogen binding. A combination of covalent and peptide bondingand disulphide bridging are particularly preferred in forming a growthfactor precursor from the modules.

The growth factor of the present invention functions after catalyticprocessing. Where the growth factor precursor comprises two B cellsurface molecule binding portions, the masking effect of the variableheavy and light chains may be in respect of both B cell surface moleculebinding portions or only one B cell surface molecule binding portion.Where the growth factor precursor molecule comprises only one B cellsurface molecule binding portion then a multimerizing inducing unit ormultimer forming portion may also be included in order to form multimersof the B cell surface molecule binding portion of the growth factor.

In a related aspect, the present invention provides a growth factorprecursor comprising a recombinant polypeptide chain or a moleculehaving modular peptide components or a synthetic equivalent thereofwherein said polypeptide chain or modular peptide molecule comprises atleast one B cell surface molecule binding portion, at least one T cellsurface molecule binding portion capable of providing T cell dependenthelp to a B cell, an antigen cleavable by a catalytic antibody and apeptide portion comprising domains from both a variable heavy chain anda variable light chain of an immunoglobulin and wherein said variableheavy chain and variable light chain domains in the growth factorprecursor, associate together by intra- and/or inter-domain bonding andsubstantially prevent the at least one B cell surface molecule bindingportion from interacting with a B cell surface molecule such that uponcleavage of said antigen by a catalytic antibody, the peptide comprisingsaid variable heavy chain and variable light chain domain permits the atleast one B cell surface molecule binding portion to interact with a Bcell surface molecule.

The T cell surface molecule binding portion provides T cell dependenthelp for the B cell. The T cell surface molecule binding portion ispreferably part of the growth factor precursor but may alternatively beexogenously supplied. An example of an exogenously supplied portionhaving T cell dependent help from a B cell is anti-CD40L antibodies orfunctional equivalents thereof.

In a further aspect of the present invention, the multimizing inducingportion comprises a signal peptide such as from the outer membraneprotein A (ompA) or a functional equivalent or derivative thereof linkedpreferably to the C-terminal portion of the growth factor.

In a particularly preferred embodiment, the B cell surface moleculebinding portions comprises a B cell surface binding portion such as a Bcell surface immunoglobulin although the present invention extends to arange of B cell surface molecules the binding, interaction and/orcross-linking of which leads to or facilitates B cell mitogenesis.

The present invention further contemplates a composition of mattercapable of inducing B cell mitogenesis of a catalytic B cell aftercatalytic processing said composition of matter comprising componentsselected from:

-   (i) a recombinant or synthetic molecule capable of inducing a B cell    surface molecule binding portion in multimeric form;-   (ii) a recombinant or synthetic molecule of (i) comprising a further    portion providing a T cell surface molecule binding portion; and-   (iii) separate compositions mixed prior to use or used sequentially    or simultaneously comprising in a first composition a component    having a B cell surface molecule binding portion and in a second    composition a molecule capable of providing a T cell surface    molecule binding portion;    said composition of matter further comprising a recombinant or    synthetic B cell surface molecule binding portion masked by    components of a variable heavy chain domain and a variable light    chain domain which variable heavy and light chains are associated    together by intra- and/or inter-domain bonding.

In a related embodiment, the present invention is directed to acomposition of matter capable of inducing B cell mitogenesis ofcatalytic B cells after catalytic processing said composition of mattercomprising components selected from:

-   (i) a recombinant or synthetic molecule comprising a B cell surface    molecule binding portion;-   (ii) a recombinant or synthetic molecule comprising a B cell surface    molecule binding portion and a signal peptide linked to the    C-terminal portion of the B cell surface molecule binding portion;-   (ii) a recombinant or synthetic molecule of (i) or (ii) comprising a    further portion providing a T cell surface molecule binding portion;    and-   (iv) separate compositions mixed prior to use or used sequentially    or simultaneously comprising in a first composition a component    having a B cell surface molecule binding portion and in a second    composition a molecule capable of providing a T cell surface    molecule binding portion;    said composition of matter further comprising a recombinant or    synthetic B cell surface molecule binding portion masked by    components of a variable heavy chain domain and a variable light    chain domain which variable heavy and light chains are associated    together by intra- and/or inter-domain bonding.

Preferably, for example to facilitate cross-linking of B cell surfacemolecules to induce mitogenesis (i.e. activation and proliferation), thegrowth factor comprises at least two B cell surface molecule bindingportions. Alternatively, where the growth factor is present inmultimeric form or is capable of being presented in multimeric form, themolecule may comprise a single B cell surface molecule binding portion.

The presentation of a T cell surface molecule binding portion on thesurface of a B cell allows for B cell mitogenesis. The term “B cellmitogenesis” is used herein in its broadest context and includes B cellactivation and proliferation, clonal expansion, affinity maturationand/or antibody secretion as well as growth and differentiation.

In accordance with the present invention, a multimer comprises two ormore growth factor molecules or a precursor thereof. Examples ofportions inducing multimerisation include but are not limited to anantibody, a region facilitating formation of cross-linked molecules or asignal peptide. Cross-linkage in this context includes any interactionthat provides bonding adequate to lead to multimer formation includingbut not limited to covalent linkage, ionic linkage, lattice association,ionic bridges, salt bridges and non-specific molecular association. Aparticularly preferred embodiment of the present invention is directedto the use of a signal peptide such as the signal peptide of ompA[Skerra, Gene, 151: 131-135, 1994] or a functional derivative thereof. A“functional derivative” in this context is a mutant or derivative of theompA signal peptide (or its functional equivalent) which still permitsmultimer formation of the growth factor.

An example of a suitable B cell surface molecule binding portion isprotein L from Peptostreptococcus magnus. Protein L has fiveimmunoglobulin-binding domains. Other immunoglobulin binding moleculesinclude protein A, protein G and protein H. The present invention,however, extends to any molecule capable of binding to a B cell surfacecomponent including, for example, a ligand of a B cell receptor.

The portion of the recombinant or synthetic molecule defining a T cellsurface molecule binding portion is presented to a preferably alreadyprimed T cell to induce B cell proliferation and affinity maturation ofan antibody in the germinal centre. This is generally accompanied byimmunoglobulin class switching and antibody secretion into the serum.Generally, the T cell surface molecule binding portion is internalisedwithin the B cell and presented on major histocompatibility complex(MHC) class II.

An example of a T cell surface molecule binding portion is from hen egglysozyme (HEL) [Altuvia et al, Molecular Immunology, 31: 1-19, 1994] oris a derivative thereof such as a peptide comprising amino acids 42 to62 from HEL or a homologue or analog thereof. This T cell surfacemolecule binding portion is recognised by the T cell receptor (TCR) ofHEL specific T cells when presented by an antigen presenting cell (APC)on the MHC class II molecule H-2A^(K) in mice or other MHC class IImolecules or their equivalents in other mammals such as humans. Examplesof other T cell surface molecule binding portions include but are notlimited to tetanus toxoid, ovalbumin, malarial antigens as well as otherregions of HEL. One skilled in the art would readily be able to selectan appropriate T cell surface molecule binding portion.

In an alternative embodiment, the portion providing the T cell surfacemolecule binding portion functions like a T cell epitope. An example ofsuch a portion is an anti-CD40L antibody.

As stated above, the B cell surface molecule binding portions induce Bcell activation and blast formation. The internalisation and processingof the growth factor leads to the presentation of the antigen on MHC II.T cell recognition of MHC II with the antigen signals the activated Bcell to proliferate and undergo antibody class switching and secretion.

The mitogenic growth factor of the present invention is most useful ingenerating antibodies of desired catalytic specificity when, in aprecursor form, it selects “catalytic” B cells. The precursor growthfactor comprises a target antigen to which a catalytic antibody issought and contains components which mask antigen-independent clonalexpansion of B cells. Upon cleavage of the antigen by a selected B cellsurface immunoglobulin, the growth factor can induce B cell mitogenesis.

In effect, then B cells are selected on the catalytic activity of theirsurface immunoglobulin rather than on their binding to a transitionstate analog. This allows for affinity maturation in the germinalcentres and ensures “catalytic-maturation” to obtain the highestenzymatic turn-over rate possible in vivo. This aspect of the presentinvention is achieved by designing growth factor precursor shielded andsubstantially inactive until released through cleavage by a catalyticantibody on a B cell surface. The term “cleavage” in this context is notlimiting to the breaking of bonds but includes an interaction adequateto remove or reduce shielding of the B cell growth factor.

The liberated growth factor activates the catalytic B cell via the Bcell surface molecule binding portion domains. The growth factor is theninternalised and processed analogous to a normal antigen. Intracellularprocessing permits the T cell surface molecule binding portion beingpresented on the B cell surface and this leads to T cell dependentclonal expansion of the B cell as well as catalytic maturation andsecretion of the catalytic antibody. The catalytic antibodies can thenbe detected in serum and “catalytic” B cells can be recovered bystandard techniques.

The antigen according to this aspect of the present invention is anyantigen to which a catalytic antibody is sought. Examples includecytokines such as but not limited to tumor necrosis factor (TNF), aninterleukin (IL) such as IL-1 to IL-15, interferons (IFN) such as IFNα,IFNβ or IFNγ, colony-stimulating factors (CSF) such as granulocytecolony-stimulating factor (G-CSF), granulocyte-macrophasecolony-stimulation factor (GM-CSF), blood factors such as Factor vIII,erythropoietin and haemopoietin, cancer antigens, docking receptors frompathogenic viruses such as HIV, influenza virus or a hepatitis virus(eg. HEP A, HEP B, HEP C or HEP E) and amyloid plaques such as inAlzheimer's disease patients or myeloma patients. More particularly, inthe case of TNF, proteolytic inactivation of TNF would be useful in thetreatment of rheumatoid arthritis and toxic shock syndrome. By targetingviral docking receptors, pathogenic viruses such as HIV, hepatitisviruses and influenza viruses are rendered effectively inactive.Catalytic antibodies will also be useful in the clearance of amyloidplaques in Alzheimer's disease or myeloma disease patients. TargetingIgE, for example, may provide a mechanism for treating inflammatoryconditions such as asthma.

The catalytic antibodies of the present invention may also be useful indetoxifying drugs such as drugs consumed by an individual. For example,the effects of cannabis or heroin or other drugs could be treated in anindividual by the administration of catalytic antibodies directed to theactive components of those drugs (Mets et al. Proc. Natl. Acad. Sci. USA95: 10176-10181, 1998). Furthermore, catalytic antibodies may be usefulin the treatment of autoimmune and inflammatory disease conditions suchas by targeting autoimmune antibodies. Catalytic antibodies also have ause in environmental and other industrial situations and could bedirected to environmental pollutants such as petroleum products andplastics. In all these situations, suitable antigens would be selectedand incorporated into the growth factor precursor of the presentinvention.

In a related aspect of the present invention, the “antigen” portion ofthe growth factor precursor can be mimicked by a target site such as anamino acid linker sequence comprising a protease cleavage site. Examplesinclude an amino acid linker sequence comprising the tabacco etch virus(TEV) protease cleavage site. More particularly, in the case of a TEVprotease cleavage site, cleaving of the amino acid linker sequence bythe TEV protease would be useful for producing characteristics similarto those of a catalytic antibody. This provides a useful model systemfor developing growth factor molecules.

The growth factor precursor enables an antigen to be recognised by a Bcell via a growth factor capable of inducing B cell mitogenesis. Thegrowth factor is in “precursor” form until cleavage of all or part ofthe antigen. It is important, however, that the B cell surface moleculebinding portions be “masked” until catalytic B cells induce cleavage ofthe target antigen and exposure of the B cell surface molecule bindingportions. Masking is provided by molecules capable of binding to orotherwise associating with the B cell surface molecule binding portion.In a particularly preferred embodiment, the masking molecules are all ora portion of the variable heavy chain domain and variable light chaindomain of an immunoglobulin.

In a particularly preferred embodiment, a fragment comprising a variableheavy and light chain (Fv domains) is employed which is a single chain(sc) and/or disulphide stabilized (ds). The scdsFV fragment isconveniently obtainable from plasmacytoma McPc603, described in (Freundet al. Biochemistry, 33: 3296-3303, 1994). The variable light and heavychain regions are preferably present as a single amino acid sequence.The regions fold and associate together by inter-domain attractiveforces. Intra-domain attractive forces may also be involved. Preferably,the intra- and inter-domain attractive forces are disulphide bonds butthe present invention extends to other forces capable of stabilising thedomains such that they fold over or are in close proximity to at leastone B cell surface molecule binding portion thus preventing B cellsurface molecule binding portion interaction with a B cell surfacemolecule. Reference to inter- and intra-domain bonding means bondingwith the polypeptide chain of the growth factor precursor and not tobonding between different polypeptide chains.

Accordingly, another aspect of the present invention is directed to agrowth factor precursor comprising a recombinant polypeptide chain or amolecule having modular peptide components or a synthetic equivalentthereof wherein said polypeptide chain or modular peptide moleculecomprises at least one B cell surface molecule binding portion, at leastone T cell surface molecule binding portion capable of providing T celldependent help to a B cell, an antigen cleavable by a catalytic antibodyand a peptide portion comprising domains from both a variable heavychain and a variable light chain of an immunoglobulin and wherein saidvariable heavy chain and variable light chain domains in the growthfactor precursor, associate together by intra- and/or inter-domainbonding and substantially prevent the at least one B cell surfacemolecule binding portion from interacting with a B cell surface moleculesuch that upon cleavage of said antigen by a catalytic antibody, thepeptide comprising said variable heavy chain and variable light chaindomain permits the at least one B cell surface molecule binding portionto interact with a B cell surface molecule.

In a related embodiment, the present invention provides a growth factorprecursor comprising a recombinant polypeptide chain or a moleculehaving modular peptide components or a synthetic equivalent thereofwherein said polypeptide chain or modular peptide molecule comprises atleast one B cell surface molecule binding portion, at least one T cellsurface molecule binding portion capable of providing T cell dependenthelp to a B cell, an antigen cleavable by a catalytic antibody and apeptide portion comprising domains from both a variable heavy chain anda variable light chain of an immunoglobulin and wherein said variableheavy chain and variable light chain domains in the growth factorprecursor associate together by intra- and/or inter-domain-bonding andsubstantially prevent the at least one B cell surface molecule bindingportion from interacting with a B cell surface molecule such that uponcleavage of said antigen by a catalytic antibody, the peptide comprisingsaid variable heavy chain and variable light chain domain permits the atleast one B cell surface molecule binding portion to interact with a Bcell surface molecule wherein if said growth factor precursor comprisesa single B cell surface molecule-binding portion, then the growth factorprecursor further comprises a multimerising inducing element.

Another aspect of the present invention provides a growth factorprecursor comprising a recombinant polypeptide chain or a moleculehaving modular peptide components or a synthetic equivalent thereofwherein said polypeptide chain or modular peptide molecule comprises atleast two B cell surface molecule binding portions, an antigen cleavableby a catalytic antibody and a peptide portion comprising domains fromboth a variable heavy chain and a variable light chain such that in thegrowth factor precursor, these variable chain components associatetogether by intra- and/or inter-domain disulphide bridges and, whenassociated together, substantially prevent at least one of the B cellsurface molecule binding portions from interacting with a B cell surfaceligand for said epitope wherein upon cleavage of said antigen by acatalytic antibody, the at least two B cell surface molecule bindingportions induce activation and proliferation of a B cell expressing saidcatalytic antibody.

A particularly useful masking molecule is derived from the variableheavy and light chain of McPc603. The latter molecule is expressed inthe periplasmic space of DH10B and can be purified on an L-column. Thevariable heavy and light chain components is preferably present on asingle peptide chain.

In a particularly preferred embodiment, the recombinant or syntheticgrowth factor precursor substantially prevents binding of at least one Bcell surface molecule binding portion to a cognate B cell surfaceimmunoglobulin thereby preventing B cell activation by havingimmunoglobulin peptide(s) or chemical equivalents thereof linked, fusedor otherwise associated with the growth factor precursor to facilitatemasking of the B cell activating effects of the growth factor. In aparticularly preferred embodiment, the precursor comprises an antigen towhich a catalytic antibody is sought and portions capable or masking a Bcell surface molecule binding portion on the growth factor precursor.The precursor preferably contains domains for variable heavy and lightchain components which associate together and exhibit inter- andintra-domain disulphide bridges.

Generally, the immunoglobulin molecules which bind to the B cell surfacemolecule binding portion of the growth factor are linked to theN-terminal and/or C-terminal portions of the growth factor. For example,one particularly preferred embodiment of the present invention providesa growth factor precursor comprising the structure:

I′AX₁[X₂]_(d)[X₃]_(a)[A]_(r)I″

wherein:X₁ and X₃ are B cell surface molecule binding portions;d is 0 or l or >l;a is 0 or 1 or >1;I′ and I″ are either both present or only one is present and they may bethe same or different and each is a blocking reagent for X₁ and/or X₃such as a variable heavy and light chain or a sc-ds-Fv molecule;A is the target antigen for which a catalytic antibody is sought;X₂ is an entity providing T cell dependent help to a B cell; andr is 0, 1 or >1,wherein a catalytic antibody on the surface of said B cell is capable ofcleaving all or part of A from said recombinant or synthetic moleculeresulting in the molecule [A′]X₁X₂[X₃]_(a)[A′] wherein A′ is optionallypresent and is a portion of A after cleavage with the catalytic antibodywherein said resulting molecule is capable of inducing T cell dependentB cell mitogenesis of the B cell to which X₁ and X₃ bind.

The molecular components of I′AX₁X₂X₃AI″ may be in any sequence order.

In another embodiment, the I′AX₁X₂X₃AI″ molecule or part thereof may bein multimeric form. This is particularly the case when all or part ofthe molecule includes a multimerisation component (M) such as but notlimited to the signal peptide of ompA. The monomeric units may be boundor otherwise associated together by any number of binding means such ascontemplated above including covalent bonding, salt bridges, disulphidebridges and hydrophobic interactions amongst many others. Depending onthe extent of multimerisation, this may impair the masking ability of Bcell surface molecule binding domains of the growth factor and someantigen-independent clonal expansion may occur. This may not be toodisadvantageous where there is at least some catalytic antibodydependent B cell mitogenesis.

According to this embodiment, there is provided a growth factorprecursor comprising the structure:

[I′AX₁[X₂′]_(o)[X₂X₃[A]_(p)I″]_(n)]_(m)

wherein:I′ and I″ are both present or only one is present and each is a blockingreagent for X₁ and/or X₃ such as a variable heavy or light component oran sc-ds-Fv;A is the target antigen for which a catalytic antibody is sought;X₁ and X₃ are B cell surface molecule binding portions;X₂ and X₂′ may be the same or different and each is an entity capable orproviding T cell dependent help for a B cell;o may be 0 or 1;p may be 0 or 1;n indicates the multimeric nature of the component in parentheses andmay be 0, 1 or >1;m indicates the multimeric nature of the component in parenthesis andmay be 1 or >1.

Preferably, n and m are each from about 1 to about 10,000 morepreferably from about 1 to about 1,000 and still more preferably fromabout 1 to about 200.

Preferably, if n is 0, then o is 1.

In alternative embodiments, the growth factor precursor comprises thestructure

[[I′AX₂X₃]_(n)[X₂′]_(o)[X_(t)AI″]_(m) or[[I′AX₁[X₂′]_(o)]_(n)[X₂X₃AI″]_(m)]

The exact number ascribed to n and m may not be ascertainable but themultimeric nature identified functionally or physically by size (eg.determined using HPLC or PAGE).

The present invention is now described by way of example only withreference to a particular growth factor precursor analogue. Thisanalogue is capable of mimicking a growth factor precursor but uses anenzyme sensitive molecule in place of the antigen. Such an analogue is auseful model for designing growth factor precursors.

The growth factor precursor analogue comprises modular components linkedtogether by a glycine-serine bridge referred to as [ggggs]₄. The unit ispresent four times. It is abbreviated herein “g”. Two B cell surfacemolecule binding portions, L, are linked by a g bridge to form the coreL-g-L. On the carboxy end of the B cell surface molecule bindingportion, a hexa-his Tag is linked to form: L-g-L-6×His. The N terminalend of the molecule comprises a TEV protease cleavage site to providethe molecule:

TEV-L-g-1-6×His.

The blocking or masking region is provided by a single chain moleculecomprising portion of a variable heavy chain and a variable light chainof McPc603. The variable portions associate together and are stabilisedby inter- and intra-domain disulphide bridges. These mask at least oneof the B cell surface molecule binding portions on L. The molecule mayalternatively only comprise a single L.

In the formula:

[I′AX₁[X₂′]_(o)[X₂X₃[A]_(p)I″]_(n)]_(m)

I′ and I″ may both be present or one or other is present and represent asingle amino acid sequence comprising a portion of the variable heavyand variable light chain of McPc603. Element A is the target antigen towhich a catalytic antibody is sought. Element A may be present once ortwice. Accordingly, p is 0 or 1. X₁ and X₃ are the B cell surfacemolecule binding portions. Two B cell surface molecule binding portionsare preferred but one B cell surface molecule binding portion maysurface. In one embodiment, when the growth factor precursor carries amultimerizing component such as the ompA, signal peptide then the growthfactor precursor may contain only a single epitope. In these cases, n is0. X₂ and X₂′ are T cell surface molecule binding portions providing Tcell dependent help for a B cell. If a single T cell surface moleculebinding portion is present, o is 0. Where the growth factor precursor isin multimeric form n and m are >1 and up to about 10,000 and 200,respectively. The elements may be in any order.

The growth factor precursor of the present invention may also containelements to assist in purification of the molecule. Examples include thehexa-His affinity tag and FLAG-tag.

The g bridge is preferred but the present invention extends to anylinking mechanism and is most preferably a flexible linking peptide.

In the example referred to above, TEV is the target site further TEVprotease which mimics the cleavage by a catalytic antibody.

Another aspect of the present invention contemplates a nucleic acidmolecule encoding the growth factor precursor herein described.According to this aspect of the present invention, there is provided anucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding a polypeptide chain or a moleculehaving modular peptide components or a synthetic equivalent thereofwherein said polypeptide chain or modular peptide molecule comprises atleast one B cell surface molecule binding portion, at least one T cellsurface molecule binding portion capable of providing T cell dependenthelp to a B cell, an antigen cleavable by a catalytic antibody and apeptide portion comprising domains from both a variable heavy chain anda variable light chain of an immunoglobulin and wherein said variableheavy chain and variable light chain components in the growth factorprecursor, associate together by intra- and/or inter-domain bonding and,when associated together, substantially prevent the at least one B cellsurface molecule binding portion from interacting with a B cell surfacemolecule such that upon cleavage of said antigen by a catalyticantibody, the peptide comprising said variable heavy chain and variablelight chain component permits the at least one B cell surface moleculebinding portion to interact with a

B Cell Surface Molecule.

The preferred nucleic acid molecule of the present invention encodes thegrowth factor precursor defined herein as ccMTLgL having the amino acidsequence substantially as set forth in SEQ ID NO: 16. The presentinvention further contemplates molecules having growth factor precursoractivity with an amino acid sequence with at least about 60% similarityto ccMTLgL. Alternative percentage similarities include at least about70%, at least about 80% and at least about 90% or above similarity toSEQ ID NO:16.

In a particularly preferred embodiment, the nucleic acid moleculecomprising a nucleotide sequence substantially set forth in SEQ ID NO:15 or a nucleotide sequence having at least 60% similarity thereto or anucleotide sequence capable of hybridising thereto under low stringencyconditions of 42° C.

Reference herein to a low stringency at 42° C. includes and encompassesfrom at least about 1% v/v to at least about 15% v/v formamide and fromat least about 1M to at least about 2M salt for hybridisation, and atleast about 1M to at least about 2M salt for washing conditions.Alternative stringency conditions may be applied where necessary, suchas medium stringency, which includes and encompasses from at least about16% v/v to at least about 30% v/v formamide and from at least about 0.5Mto at least about 0.9M salt for hybridisation, and at least about 0.5Mto at least about 0.9M salt for washing conditions, or high stringency,which includes and encompasses from at least about 31% v/v to at leastabout 50% v/v formamide and from at least about 0.01M to at least about0.15M salt for hybridisation, and at least about 0.01M to at least about0.15M salt for washing conditions.

The term “similarity” as used herein includes exact identity betweencompared sequences at the nucleotide or amino acid level. Where there isnon-identity at the nucleotide level, “similarity” includes differencesbetween sequences which result in different amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. Where there is non-identity atthe amino acid level, “similarity” includes amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. In a particularly preferredembodiment, nucleotide and sequence comparisons are made at the level ofidentity rather than similarity. Any number of programs are available tocompare nucleotide and amino acid sequences. Preferred programs haveregard to an appropriate alignment. One such program is Gap whichconsiders all possible alignment and gap positions and creates analignment with the largest number of matched bases and the fewest gaps.Gap uses the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443-453, 1970). Gap reads a scoring matrix that contains values forevery possible GCG symbol match. GAP is available on ANGIS (AustralianNational Genomic Information Service) at websitehttp://mel1.angis.org.au.

In a related embodiment, the present invention provides a nucleic acidmolecule encoding the growth factor precursor herein described.According to this aspect of the present invention, there is provided anucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding a polypeptide chain or a moleculehaving modular peptide components or a synthetic equivalent thereofwherein said polypeptide chain or modular peptide molecule comprises atleast one B cell surface molecule binding portion, at least one T cellsurface molecule binding portion capable of providing T cell dependenthelp to a B cell, an antigen cleavable by a catalytic antibody and apeptide portion comprising domains from both a variable heavy chain anda variable light chain of an immunoglobulin and wherein said variableheavy chain and variable light chain components in the growth factorprecursor, associate together by intra- and/or inter-domain bonding and,when associated together, substantially prevent the at least one B cellsurface molecule binding portion from interacting with a B cell surfacemolecule such that upon cleavage of said antigen by a catalyticantibody, the peptide comprising said variable heavy chain and variablelight chain component permits the at least one B cell surface moleculebinding portion to interact with a B cell surface molecule wherein ifsaid growth factor precursor comprises a single B cell surface moleculebinding portion, then the growth factor precursor further comprises amultimerising inducing element.

In another embodiment, the present invention is directed to a nucleicacid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding a polypeptide chain or a moleculehaving modular peptide components or a synthetic equivalent thereofwherein said polypeptide chain or modular peptide molecule comprises atleast one B cell surface molecule binding portion, at least one T cellsurface molecule binding portion capable of providing T cell dependenthelp to a B cell, an antigen cleavable by a catalytic antibody and apeptide portion comprising domains from both a variable heavy chain anda variable light chain of an immunoglobulin and wherein said variableheavy chain and variable light chain components in the growth factorprecursor, associate together by intra- and/or inter-domain bonding and,when associated together, substantially prevent the at least one B cellsurface molecule binding portion from interacting with a B cell surfacemolecule such that upon cleavage of said antigen by a catalyticantibody, the peptide comprising said variable heavy chain and variablelight chain component permits the at least one B cell surface moleculebinding portion to interact with a B cell surface molecule.

Preferably, the nucleic acid molecule is in form of a genetic “vaccine”.In this regard, a genetic vaccine conveniently comprises the nucleicacid molecule in, for example, a viral vector or other suitable nucleicacid transferring medium. Generally, one or more pharmaceuticallyacceptable carriers and/or diluents are also included. The geneticvaccine is introduced to cells either directly (e.g. intramuscularly),or systemically or cells are removed from an individual, the geneticvaccine introduced into the cells and then the cells are returned to theindividual or a genetically related individual. The nucleic acid in thegenetic vaccine after introduction to cells is expressed to produce thegrowth factor precursor of the present invention.

In a particularly preferred embodiment, the nucleic acid molecule in thegenetic vaccine further comprises a nucleotide sequence encoding amolecular adjuvant. Examples of suitable molecular adjuvants includeCTLA4 (Boyle et al. Nature 392: 408-411, 1998), CD40L (Lane et al. J.Exp. Med. 177: 1209-1213, 1993) and C3d (Dempsey et al. Science 27:348-350, 1996; Lou and Kohler, Naurve Biotechnology 16: 458-462, 1998).

The present invention extends to recombinant polypeptides defining thegrowth factor precursor and further comprising a molecular adjuvantattached thereto.

Upon cleavage of the growth factor precursor by a catalytic antibodyrecognising the antigen (for example, a TNF peptide portion), thecovalent linkage between the B cell surface molecule binding portion andthe variable heavy and light domains is broken. The blocking variablechains will dissociate from the B cell surface molecule binding portiondue to the relatively low affinity (˜10⁻⁷M) of individual domains foreach other. This will release the mature growth factor which can bind toand crosslink the surface immunoglobulin.

Catalytic antibodies can be detected in the serum using any number ofprocedures such as ELISA based assays and catalytic B cells may berecovered with standard hybridoma technology. Where the catalyticantibodies are from non-human animals, these can be humanised byrecombinant DNA technology and produced for therapeutical applicationsin humans. Alternatively, the antibodies may be generated in a“humanized” animal such as a humanized mouse which is transgenic for thehuman Ig loci.

The present invention contemplates derivatives of the growth factorand/or its precursor. A derivative includes a mutant, part, fragment,portion, homologue or analogue of the growth factor and/or precursor orany components thereof. Derivatives to amino acid sequences includesingle or multiple amino acid substitutions, deletions and/or additions.

Particularly useful derivatives include chemical analogues of the growthfactor precursor and/or its components. Such chemical analogues may beuseful in stabilizing the molecule for therapeutic, diagnostic andindustrial use.

Analogues of the growth factor precursor contemplated herein include,but are not limited to, modification to side chains, incorporating ofunnatural amino acids and/or their derivatives during peptide,polypeptide or protein synthesis and the use of crosslinkers and othermethods which impose conformational constraints on the proteinaceousmolecule or their analogues.

Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonicacid (TNBS); acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitisation, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids. A list of unnaturalimino acid, contemplated herein is shown in Table 1.

Crosslinkers can be used, for example, to stabilise 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety such as maleimido or dithiomoiety (SH) or carbodiimide (COOH). In addition, peptides can beconformationally constrained by, for example, incorporation of C_(α) andN_(α)-methylamino acids, introduction of double bonds between C_(α) andC_(β) atoms of amino acids and the formation of cyclic peptides oranalogues by introducing covalent bonds such as forming an amide bondbetween the N and C termini, between two side chains or between a sidechain and the N or C terminus.

The present invention further contemplates chemical analogues of thegrowth factor precursor capable of acting as antagonists or agonists ofsame. These may be useful in controlling the immunological response.Chemical analogues may not necessarily be derived from the growth factorprecursor but may share certain conformational similarities.Alternatively, chemical analogues may be specifically designed to mimiccertain physiochemical properties of the growth factor precursor.Chemical analogues may be chemically synthesised or may be detectedfollowing, for example, natural product screening of, for example,coral, soil, plants, microorganisms, marine invertebrates or seabeds.Screening of synthetic libraries is also contemplated by the presentinvention.

TABLE 1 Non-conventional Non-conventional amino acid Code amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmglncarboxylate L-N-melhylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-gluiamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-melhylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycinecarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbcethylamino)cyclopropane

Other derivatives contemplated by the present invention include a rangeof glycosylation variants from a completely unglycosylated molecule to amodified glycosylated molecule. Altered glycosylation patterns mayresult from expression of recombinant molecules in different host cells.

Still a further aspect of the present invention extends to a method forproducing catalytic antibodies to a specific antigen, said methodcomprising administering to an animal an effective amount of a growthfactor precursor comprising an antigen capable of interacting with a Bcell bound catalytic antibody said antigen linked to or otherwiseassociate with a B cell surface molecule binding portion and a portioncapable of providing T cell dependent help to a B cell. The growthfactor precursor further comprises a B cell surface molecule bindingportion masking entity such as a portion of a variable heavy and lightchain linked to the antigen.

Alternatively, the growth factor precursor may comprise a B cell surfacemolecule binding portion in multimeric form linked to an antigen forwhich a target antibody is sought. The portion providing T celldependent help is preferably a T cell surface molecule binding portionand is preferably part of the precursor. However, it may be a separateentity administered simultaneously or sequentially to an animal. Again,the B cell surface molecule binding portion is masked as above.

The present invention also provides catalytic antibodies produced by theabove method. Such catalytic antibodies may be directed to any antigensuch as but not limited to a cytokine, for example, tumor necrosisfactor (TNF), an interleukin (IL) such as IL-1 to IL-15, interferons(IFN) such as IFNα, IFNβ or IFNγ, colony-stimulating factors (CSF) suchas granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulation factor (GM-CSF), blood factors such as Factor VIII,erythropoietin and haemopoietin, cancer antigens, docking receptors frompathogenic viruses such as HIV, influenza virus or a hepatitis virus(eg. HEP A, HEP B, HEP C or HEP E) and amyloid plaques such as inAlzheimer's disease patients or myeloma patients.

The catalytic antibodies of the present invention have particulartherapeutic and diagnostic uses especially in relation to mammalian andmore particularly human disease conditions.

Accordingly, the present invention contemplates a pharmaceuticalcomposition comprising a growth factor precursor or a derivative thereofand optionally a modulator of growth factor precursor activity and oneor more pharmaceutically acceptable carriers and/or diluents. Moreparticularly, the pharmaceutical composition comprises catalyticantibodies generated by the growth factor precursor of the presentinvention. These components are hereinafter referred to as the “activeingredients”.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or sterile powders for theextemporaneous preparation of sterile injectable solutions. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol and liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils. The preventions of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimersal and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed bysterilization such as by filtration. In the case of sterile powders forthe preparation of sterile injectable solutions, the preferred methodsof preparation are vacuum drying and the freeze-drying technique whichyield a powder of the active ingredient plus any additional desiredingredient from previously sterile-filtered solution thereof.

When the active ingredients are suitably protected they may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets, or it may be incorporateddirectly with the food of the diet. For oral therapeutic administration,the active compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 5 to about 80% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions in such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 0.1 ng and 2000 mg of active compound, preferably betweenabout 0.1 μg and 1500 mg and more preferably between about 1 mg and 100mg.

The tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter: a binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such a sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen, or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry, orange or mango. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound(s) may be incorporated intosustained-release preparations and formulations.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions. These may include immunepotentiating molecules, multimer facilitating molecules andpharmaceutically active molecules chosen on the disease conditions beingtreated.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from 0.1 ng to about 2000 mg, morepreferably ranging from 0.1 μg and 1500 mg and even more preferablyranging between 1 μg and 1000 mg. Expressed in proportions, the activecompound is generally present in from about 0.5 μg to about 2000 mg/mlof carrier. In the case of compositions containing supplementary activeingredients, the dosages are determined by reference to the usual doseand manner of administration of the said ingredients.

Still another aspect of the present invention is directed to antibodiesto the growth factor precursor and its derivatives. Such antibodies maybe monoclonal or polyclonal and are independent to the catalyticantibodies selected by the precursor. The (non-catalytic) antibodies torecombinant or synthetic the growth factor precursor or its derivativesof the present invention may be useful as therapeutic agents but areparticularly useful as diagnostic agents. Antibodies may also begenerated to the catalytic antibodies generated by the growth factorprecursors. All these antibodies have particular application indiagnostic assays for the growth factor or catalytic antibody inducerthereof.

For example, specific antibodies can be used to screen for catalyticantibodies. The latter would be important, for example, as a means forscreening for levels of these antibodies in a biological fluid or forpurifying the catalytic antibodies. Techniques for the assayscontemplated herein are known in the art and include, for example,sandwich assays and ELISA.

It is within the scope of this invention to include any secondantibodies (monoclonal, polyclonal or fragments of antibodies orsynthetic antibodies) directed to the antibodies discussed above. Boththe first and second antibodies may be used in detection assays or afirst antibody may be used with a commercially availableanti-immunoglobulin antibody.

Both polyclonal and monoclonal antibodies are obtainable by immunizationwith the enzyme or protein and either type is utilizable forimmunoassays. The methods of obtaining both types of sera are well knownin the art. Polyclonal sera are less preferred but are relatively easilyprepared by injection of a suitable laboratory animal with an effectiveamount of antigen, or antigenic parts thereof, collecting serum from theanimal, and isolating specific sera by any of the known immunoabsorbenttechniques. Although antibodies produced by this method are utilizablein virtually any type of immunoassay, they are generally less favouredbecause of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularlypreferred because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are well known to those who are skilled inthe art.

Another aspect of the present invention contemplates a method fordetecting an antigen in a biological sample from a subject said methodcomprising contacting said biological sample with an antibody specificfor said antigen or its derivatives or homologues for a time and underconditions sufficient for an antibody-antigen complex to form, and thendetecting said complex. In this context, the “antigen” may be a growthfactor, its precursor, a component thereof or a catalytic antibodyinduced thereby.

The presence of antigen may be accomplished in a number of ways such asby Western blotting and ELISA procedures. A wide range of immunoassaytechniques are available as can be seen by reference to U.S. Pat. Nos.4,016,043, 4, 424,279 and 4,018,653. These, of course, includes bothsingle-site and two-site or “sandwich” assays of the non-competitivetypes, as well as in the traditional competitive binding assays. Theseassays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays andare favoured for use in the present invention. A number of variations ofthe sandwich assay technique exist, and all are intended to beencompassed by the present invention. Briefly, in a typical forwardassay, an unlabelled antibody is immobilized on a solid substrate andthe sample to be tested brought into contact with the bound molecule.After a suitable period of incubation, for a period of time sufficientto allow formation of an antibody-antigen complex, a second antibodyspecific to the antigen, labelled with a reporter molecule capable ofproducing a detectable signal is then added and incubated, allowing timesufficient for the formation of another complex ofantibody-antigen-labelled antibody. Any unreacted material is washedaway, and the presence of the antigen is determined by observation of asignal produced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof hapten. Variations on the forward assay include a simultaneous assay,in which both sample and labelled antibody are added simultaneously tothe bound antibody. These techniques are well known to those skilled inthe art, including any minor variations as will be readily apparent. Inaccordance with the present invention the sample is one which mightcontain an antigen including cell extract, supernatant fluid, tissuebiopsy or possibly serum, saliva, mucosal secretions, lymph, tissuefluid and respiratory fluid. The sample is, therefore, generally abiological sample comprising biological fluid but also extends tofermentation fluid and supernatant fluid such as from a cell culture.

In the typical forward sandwich assay, a first antibody havingspecificity for the antigen or antigenic parts thereof, is eithercovalently or passively bound to a solid surface. The solid surface istypically glass or a polymer, the most commonly used polymers beingcellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene. The solid supports may be in the form of tubes, beads,discs of microplates, or any other surface suitable for conducting animmunoassay. The binding processes are well-known in the art andgenerally consist of cross-linking covalently binding or physicallyadsorbing, the polymer-antibody complex is washed in preparation for thetest sample. An aliquot of the sample to be tested is then added to thesolid phase complex and incubated for a period of time sufficient (e.g.2-40 minutes, or overnight if more convenient) and under suitableconditions (e.g. from room temperature to about 40° C. such as 25-37°C.) to allow binding of any subunit present in the antibody. Followingthe incubation period, the antibody subunit solid phase is washed anddried and incubated with a second antibody specific for a portion of thehapten. The second antibody is linked to a reporter molecule which isused to indicate the binding of the second antibody to the hapten.

An alternative method involves immobilizing the target molecules in thebiological sample and then exposing the immobilized target to specificantibody which may or may not be labelled with a reporter molecule.Depending on the amount of target and the strength of the reportermolecule signal, a bound target may be detectable by direct labellingwith the antibody. Alternatively, a second labelled antibody, specificto the first antibody is exposed to the target-first antibody complex toform a target-first antibody-second antibody tertiary complex. Thecomplex is detected by the signal emitted by the reporter molecule.

By “reporter molecule” as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes) and chemiluminescent molecules. In the case of an enzymeimmunoassay, an enzyme is conjugated to the second antibody, generallyby means of glutaraldehyde or periodate. As will be readily recognized,however, a wide variety of different conjugation techniques exist, whichare readily available to the skilled artisan. Commonly used enzymesinclude horseradish peroxidase, glucose oxidase, beta-galactosidase andalkaline phosphatase, amongst others. The substrates to be used with thespecific enzymes are generally chosen for the production, uponhydrolysis by the corresponding enzyme, of a detectable colour change.Examples of suitable enzymes include alkaline phosphatase andperoxidase. It is also possible to employ fluorogenic substrates, whichyield a fluorescent product rather than the chromogenic substrates notedabove. In all cases, the enzyme-labelled antibody is added to the firstantibody hapten complex, allowed to bind, and then the excess reagent iswashed away. A solution containing the appropriate substrate is thenadded to the complex of antibody-antigen-antibody. The substrate willreact with the enzyme linked to the second antibody, giving aqualitative visual signal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of haptenwhich was present in the sample. “Reporter molecule” also extends to useof cell agglutination or inhibition of agglutination such as red bloodcells on latex beads, and the like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic colour visually detectable with a lightmicroscope. As in the EIA, the fluorescent labelled antibody is allowedto bind to the first antibody-hapten complex. After washing off theunbound reagent, the remaining tertiary complex is then exposed to thelight of the appropriate wavelength the fluorescence observed indicatesthe presence of the hapten of interest. Immunofluorescence and EIAtechniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotope, chemiluminescent or bioluminescentmolecules, may also be employed.

The present invention may use any number of means to clone geneticsequences encoding catalytic antibodies. For example, a phage displaylibrary potentially capable of expressing a catalytic antibody on thephage surface may be used to screen for catalysis of defined antigens.

The present invention further contemplates the use of the products ofcatalysis of a growth factor precursor to induce B cell mitogenesis togenerate catalytic antibodies to a specific antigen.

More particularly, the present invention contemplates the use of agrowth factor precursor comprising an antigen to which a catalyticantibody is sought linked, fused or otherwise associated to a B cellsurface molecule binding portion in the induction of B cell mitogenesisfollowing catalytic cleavage of all or part of said antigen.

Still another embodiment of the present invention contemplates the useof an antigen linked, fused or otherwise associate to a B cell surfacemolecule binding portion in the manufacture of a growth factor precursorto induce B cell mitogenesis following catalytic cleavage of all or partof said antigen.

The present invention is further described by the following non-limitingexamples.

Example 1 Generation of LHL from Synthetic Oligonucleotides

LHL was generated from three overlapping synthetic oligos, a 115mer, a116mer and a 105mer, using the proofreading DNA polymerase Pfu in two 20cycle PCR reactions. The two PCR products (290 bp and 200 bp) werepurified and blunt end cloned into the expression vector pASK75. Thesequence was verified by automated sequencing. All subsequent PCRs weredone in a similar fashion as described in the literature. The nucleotideand corresponding amino acid sequence for LHL is shown in SEQ ID NO: 1and SEQ ID NO:2 respectively.

Example 2 Expression of LHL in E. coli and Purification Over a Human IgG(huIgG) Affinity Column

The expression vector pASK75 directs protein expression via the ompAsignal peptide into the periplasm of E. coli. Protein expression wasinduced with 200 ng/ml anhydrotetracycline for 16 hrs in midlog E. coliDH10B cultures. Cells were lysed and soluble LHL purified (>95%) over ahuIgG affinity column. Extensive washes with 0.5% v/v Triton X-100 wereperformed on the affinity column in order to eliminate endotoxins fromthe preparations. Expression levels were estimated at 200 mg per litreof culture.

Example 3 Generation of an LHL Protein Carrying the N-Terminal FlagEpitope and the C-Terminal Strep-Tag

A form of LHL (referred to herein as “LHL.seq”) was generated by PCRcontaining the FLAG epitope at its N-terminus and the so calledstrep-tag at its C-terminus. The nucleotide and corresponding amino acidsequence for LHL.seq is shown in SEQ ID NO:7 and SEQ ID NO:8,respectively. The FLAG epitope comprises the amino acids DYKDDDDK (SEQID NO:9) and the strep-tag the amino acids AWRHPQFGG (SEQ ID NO: 14).The FLAG epitope is recognised by several anti-FLAG monoclonalantibodies and the strep-tag by streptavidin. The strep-tag was used forpurification of LHL.seq over a streptavidin column. LHL.seq was washedwith 0.5% v/v Triton X-100, Tween20 and PBS while bound to the column inorder to minimise endotoxin levels. LHL.seq was eluted with either 100mM glycine pH2.0 or with 1 mg/ml diaminobiotin in PBS. In this methodLHL.seq was not purified on the basis of binding immunoglobulin, therebyeliminating potential contamination of other unknown bacterial proteinswhich also bind immunoglobulins. The biological activity of LHL.seq,however, remained identical to that of LHL. The FLAG-epitope was addedto the N-terminus in order to facilitate the secretion of LHL.seq intothe periplasmic space. As in previous expression studies, this wasunsuccessful and LHL.seq needed to be purified from total bacteriallysate. As a result of this, the ompA signal peptide is not removed,which in turn led to formation of LHL.seq multimers.

Example 4 Mitogenic Activity of LHL on B Cells

Mitogenic activity of LHL on B cells was tested in overnight cultures ofsplenocytes and mesenteric lymphocytes as well as on purified B cells.The activation status of B cells was analysed by FACS, examining B cellsize and induction of B7-2 surface expression. LHL's activation potencyis similar to LPS (10 μg/ml), a bacterial mitogenic lipopolysaccharideand anti-IgM antibody (25 μg/ml), which crosslinks surface IgM. Theresults have been independently obtained in several different mousestrain e.g. B10.A(4R), CBA, C3H/HeJ and BALB/c. B cells showed a cleardose response to LHL when titrated in 5-fold dilutions (25 μg/ml to 1.6ng/ml) in the activation assay. Parallel experiments analysing the Tcell activation status within the same cultures demonstrated that LHLhas no effect on T cells. T cells did not show any blast formation nordid they upregulate activation markers, e.g. IL-2 receptor alpha chain(CD25).

Example 5 Blocking of LHL Mitogenicity by HuIgG

In the same experiments, soluble hulgG (500 μg/ml) which binds to the Ldomains was used to specifically block the activity of LHL. Theseresults rule out that B cell activation was due to a contamination ofthe bacterially produced LHL with endotoxins.

Example 6 Processing of LHL by B Cells and Presentation Of the H Epitopeto the HEL-Specific Hybridoma 3A9

Splenocytes or mesenteric lymphocytes were cocultured with the T cellhybridoma 3A9 in the presence of LHL. 3A9 is specific for the HELpeptide 52-61aa presented on MHC II H-2A^(K). Upon recognition of thispeptide, 3A9 secretes IL-2. L-2 production was measured in a bio assaywhich evaluates the proliferation of an IL-2 dependent cell line (CTLL)on the basis of ³H-thymidine incorporation during DNA synthesis.Presentation of H to 3A9 by B cells was clearly demonstrated by theproliferation of the CTLL and could be specifically blocked with huIgG.

Example 7 Generation of the Variable (V)-Kappa Light Chain According tothe Human Len Protein Sequence

The amino acid sequence of the gene encoding the human myeloma proteinLEN was used to generate a variable kappa light chain. This human kappalight chain protein (hereinafter referred to as “kappa”) is soluble atrelatively high concentrations and has been shown to bind protein L.Kappa was generated from synthetic oligonucleotides by PCR. Tofacilitate protein purification, a FLAG epitope was added to theN-terminus and a strep-tag to the C-terminus. The nucleotide and aminoacid sequence of kappa is shown in SEQ ID NO: 10 and 11, respectively.

Example 8 Expression of Kappa in E. coli DH10B

Kappa was cloned into pASK75, allowing inducible expression of kappainto the periplasmic space of E. coli. Expression was induced inlogarithmically growing cultures of E. coli strain DH10B cells with 400ng/ml of anhydro-tetracycline for >4 hrs.

Example 9 Purification of Kappa Protein from the Periplasm of DH10B

Cultures were spun down and resuspended in a buffer containing 400 mMsucrose on ice.

After 20 min cells were pelleted. Kappa was then purified over ananti-FLAG and/or streptavidin column from the periplasmic fraction.

Example 10 Confirmation of Proper Folding of Kappa after Purification

The proper folding of kappa was demonstrated by its capacity to bindLHL. Kappa was bound to the streptavidin column via its strep-tag. Thiskappa-loaded column was then shown to bind LHL. The non strep-tagcarrying LHL did not bind to the streptavidin column alone.

Example 11 Generation of TLHL

TLHL was generated from LHL, kappa and synthetic oligonucleotidesencoding a linker connecting kappa and LHL by PCR. The linker containedan amino acid sequence corresponding to the tobacco etch virus (TEV)protease recognition/cleavage site. All components were cloned intopASK75 resulting in the following protein sequence:FLAG-kappa-linker-TEV-LHL-streptag. Potentially, TLHL could show similarcharacteristics as CATAB, since one kappa binding site is blocked andtwo are required for surface immunoglobulin cross-linking. Thenucleotide and amino acid sequences of TLHL are shown in SEQ ID NO:5 andSEQ ID NO:6, respectively.

Example 12 Expression of TLHL in Dh10B

TLHL expression was induced in logarithmically growing cultures byaddition of 400 ng/ml anhydro-tetracycline for >4 hrs. TLHL was notsecreted into the periplasmic space and caused some cell lysis afterinduction.

Example 13 Purification of TLHL from Total Bacterial Lysate

TLHL was purified via its strep-tag over a streptavidin column fromtotal bacterial lysate. Endotoxin levels were reduced using the washingprotocol earlier described.

Example 14 Cleavage of TLHL into “T” and “LHL” with TEV

TLHL was designed so that the kappa portion of the protein could becleaved off by the TEV protease. The TEV cleavage would generate twopolypeptides, each of 172 amino acids. The identical size of the proteinfragments is due to TLHL not being secreted into the periplasmic spaceof E. coli and, therefore, retaining the ompA signal peptide. Incubationof TLHL with the TEV protease in PBS at room temperature or at 4° C.produced therefore, a 19 kD band on an SDS-PAGE gel.

Example 15 Assembly of CATAB-TEV from TLHL and Kappa by PCR

CATAB-TEV is assembled from TLHL and kappa by PCR. The TLHL and kappacan be linked by different peptides, for example, TNF amino acids 1-31,that are potential target sites for proteolytic antibodies. In thiscase, the linker includes a recognition sequence for the tobacco etchvirus (TEV) protease which allows the generation of LHL from CATAB-TEVin vitro. The nucleotide and corresponding amino acid sequences ofCATAB-TEV are shown in SEQ ID NO:3 and SEQ ID NO:4.

Example 16 Expression of CATAB in Dh10B and Purification Over aStreptavidin Affinity Column Via Strep-Tag

CATAB-TEV is expressed and purified in the same way as TLHL (see above).

Example 17 Demonstration of Non-Mitogenic Activity of CATAB-TEV on BCells

CATAB-TEV is tested in the already established B cell assays which areused to analyse the mitogenic activity of LHL and LHL.seq.

Example 18 Revelation of the Mitogenic Activity of CATAB by ProteolyticCleavage with TEV Protease

Digestion of CATAB-TEV with the site specific protease from TEV cleavesthe covalent bond between LHL and the kappa domains. This cleavagegenerates the mitogenic compound LHL which is tested in the standardisedB cell activation assays.

Example 19 Usage of CATAB in Several Mouse Strains of the K-Haplotype

Several mouse strains are immunised by different routes ofadministration, e.g. intra-splenic, in order to elicit a catalyticantibody response in vivo. The gld and lpr mutant strains are used asthey have been shown to have a relatively high incidence of naturallyoccurring catalytic auto-antibodies, e.g. antibodies with DNAseactivity.

Example 20 Detection of CATAB Specific Catalytic Antibodies From theSerum

Serum antibodies from immunised mice are purified for example on a LHLaffinity column. Purified antibodies may be incubated with ¹²⁵I-labelledCATAB and the proteolytic cleavage is evaluated on PAGE gels. Inaddition, streptavidin may be used to immobilise CATAB via itsC-terminal strep-tag on 96 well ELISA plates. Immobilised CATAB isproteolytically cleaved by incubation with purified catalytic serumantibodies and an N-terminal affinity tag, e.g. flag epitope, is lost.This loss is detected in a sandwich ELISA assay using horse radishperoxidase (HRPO) conjugated antibodies. B cells producing catalyticantibodies can be recovered by standard hybridoma techniques and thecatalytic antibodies can be humanised by recombinant DNA technology. Forexample, “human” antibodies can be derived from humanized mice.

Example 21 LHL.seq Induced B7-1 Expression

LHL.seq % vas tested for its ability to activate B cells as compared tostimulation with anti-IgM and anti-kappa. Activation status was measuredby the induction of cell surface expression of the activation markersB7-1 and B7-2 and by entry of B cells into cell cycle. Levels ofexpression of B7-1 and B7-2 were determined by flow cytometry (FACS)with fluorescence-labelled monoclonal antibodies while entry into cellcycle was monitored by an increase in cell size by Forward Light Scatter(FSC).

The method employed was as follows. Mesenteric lymphnode cells fromC3H/HeJ mice were centrifuged in Nycodenz (1.091 g/cm³) to remove deadcells and red blood cells (rbc). This was followed by 1 hour adherenceon plastic at 37° C. to remove adherent cells such as macrophages. Lymphnode cells were stimulated in triplicate cultures 3×10⁵/well in flatbottom 96-well plates in complete RPMI+10% FCS medium at 37° C. for 1-3days. Upregulation of activation markers on B cells was monitored bygating on B220⁺Thy1⁻ cells to identify B cells. Stimulation with LPS (20μg/ml), polyclonal F(ab)₂ anti-IgM antibodies (20 μg/ml) and anti-kappaantibodies (10 μg/ml) were included as controls. LHL.seq was used at 1μg/ml. C3H/HeJ mice were used as source of lymphocytes since thisparticular mouse strain is non-responsive to LPS. The use of this strainin combination with the LPS control effectively precludes thepossibility that B cell stimulation induced by LHL.seq were, due to LPS(endotoxin) contamination of the bacterially expressed proteins.

FACS analysis showed that this two day stimulation of C3H/HeJ lymph nodecells with LPS did not result in B cell activation whereas stimulationwith either anti-IgM antibodies, anti-kappa antibodies or LHL.seq did asmeasured by an increased FSC and upregulation of B7-2. Thecharacteristic potency of LHL.seq is demonstrated by the stronginduction of B7-1 expression after incubation. Anti-IgM induces B7-1 onday 2-3 of stimulation.

Example 22 LHL.seq Induced MHC Class II

LHL.seq was compared in its potential to ensure proper upregulation ofMHC class II on stimulated B cells. Anti-IgM antibodies (20 μg/ml) aswell as LHL seq (1 μg/ml) blocked with huIgG (500 μg/ml) were includedas controls. The method used was as described in Example 21.

Upregulation of MHC Class II molecules on B cells is a prerequisite toreceive T cell help in vivo.

Overnight stimulation of C3H/HeJ lymph node cells with anti-IgMantibodies as well as LHL.seq did result in increased FSC andupregulation of MHC class II. LHL.seq's activities were completelyblocked by addition of 500 μg/ml hulgG to the cultures.

Example 23 LHL.seq Induced Proliferation in a Dose Dependent Fashion

Serial dilutions of LHL.seq were used to stimulate B cell proliferation.The experiment demonstrated that LHL.seq's biological properties aresimilar to conventional B cell mitogens like anti-IgM antibodies. Thus,dose-response curves for stimulation of either mesenteric lymphnodecells from C3H/HeJ and splenocytes from CBA/J were obtained.

Example 24 TLHL Induced B Cell Activation

LHL.seq, TLHL and TEV-cleaved TLHL were tested for their ability toactivate B cells as measured by the induction of cell surface expressionof the activation markers B7-1 (CD86) and B7-2 (CD80) and by entry of Bcells into cell cycle. Levels of expression of B7-1 and B7-2 weredetermined by flow cytometry (FACS) with fluorescence-labelledmonoclonal antibodies while proliferation was monitored by an increasein cell size by Forward Light Scatter (FSC) and by ³H-thymidine-uptakeassays.

The method employed as described in Example 21.

Overnight stimulation of C3H/HeJ lymph node cells with LPS did notresult in B cell activation whereas stimulation with either anti-IgMantibodies or LHL.seq did as measured by an increased FSC andupregulation of B7-2. The characteristic potency of LHL.seq isdemonstrated by the strong induction of B7-1 expression after overnightincubation. Anti-IgM induces B7-1 on day 2-3 of stimulation.

TLHL, however, activated B cells to the same extent as LHL.seq. This wasunexpected since it was presumed that blocking one L domain with acovalently linked kappa would prevent crosslinking of immunoglobulin onthe B cell surface. Prevention of crosslinking should result in no orsignificantly lower B cell activation than that achieved with equalamounts of LHL.seq. TEV-cleaved TLHL, which results in omp-kappa (seebelow) plus the LHL.seq part, gave much lower B cell activation thanuncleaved TLHL as indicated by less B7-1 and B7-2 upregulation and lowerFSC increase.

Splenocytes from CBA/J mice were centrifuged in Nycodenz (1.091 g/cm³)to remove dead cells and rbc. This was followed by 1 hour adherence onplastic at 37° C. to remove adherent cells. Splenocytes were thenstimulated in triplicate cultures at 2×10⁵/well in flat bottom 96-wellplates in complete RPMI+10% v/v FCS medium at 37° C. for 2 days. Cellswere pulsed for the last 6 hours with ³H-thymidine. DNA was thenharvested onto glassfibre filters and incorporation of ³H-thymidine wasmeasured in a β-counter.

The results obtained by FACS analysis were confirmed by theproliferation data; TLHL and LHL.seq induced equivalent B cellproliferation while TEV-cleaved TLHL was about 70% less potent.

Example 25 TEV-Cleaved TLHL Stimulation Data Confirm OMP InducedMultimerisation

The B cell activation data lead the inventors to the conclusion thatboth LHL, LHL.seq and TLHL exist in solution as multimeric molecules.While dimeric or oligomeric immunoglobulin-binding molecules such asanti-IgM antibodies induce B cell activation, multimers such asanti-Igb-dextran result in a significantly higher degree of B cellactivation. This is also the case with LHL, LHL.seq and TLHL in theabove experiments as demonstrated by the extensive upregulation of B7-1after overnight culture. The multimerisation is facilitated by the ompAsignal peptide (omp). It has been published by others that the ompAsignal peptide forms multimers in aqueous solution. Evidence for LHL,LHL.seq and TLHL aggregation has also been obtained in HPLC studies.

A new recombinant LHL.seq protein lacking the ompA signal peptide,called LHL-omp, was engineered which also confirms these conclusions(see below).

Example 26 TLHL Multimerisation Overcomes “Kappa-Blocking”

Although one ‘L’ domain should be blocked by kappa in TLHL, themultimerisation mediated by the omp allows several free ‘L’ domains toexist in one multimeric molecule [TLHL]_(n). This will lead to extensivesIg crosslinking and full B cell activation as demonstrated.

Example 27 Generation and Analysis of LHL-OMP

LHL-omp was generated from LHL.seq via PCR with the proofreadingpolymerase Pfu eliminating the ompA signal sequence.

Example 28 Affinity Column Purification of LHL-OMP

Although LHL-omp contains a Strep-tag, it could not be purified via theStreptavidin column using the standard protocol, indicating a loweravidity to the column matrix than that of LHL.seq. This lower avidityconfirms the multimerisation of LHL.seq via omp, being the onlydifference between LHL.seq and LHL-omp. In agreement with this LHL-ompwas readily purified over a huIgG affinity column.

Example 29 LHL-OMP Induced B Cell Activation

The ability of LHL-omp to induce B cell activation was assessed byincubating splenocytes from C3H/HeJ mice for varying periods of timebefore analysing B7-1 and B7-2 expression levels on B cells as outlinedabove. The progression of B cells into cell cycle was monitored by FACSand proliferation assays.

Cells were prepared and cultured as described above. LPS (20 μg/ml) andanti-IgM (20 μg/ml) were used as controls.

Stimulation of C3H/HeJ splenocytes with LPS did not result in detectableB cell activation whereas treatment with either anti-IgM antibodies orLHL.seq induced B cell activation during overnight culture; increasedFSC and B7-2 upregulation for anti-IgM antibodies and increased FSC andB7-1 and B7-2 expression for LHL.seq. LHL-omp, used at 2 μg/ml, was lesspotent than LHL.seq in inducing upregulation of B7-1, B7-2 and blastingof B cells, as indicated by the FSC profile. The unchanged FSC profileindicated that LHL-omp did not induce B cell proliferation. This wasconfirmed in proliferation assays.

B cells were stimulated simultaneously with LHL-omp and anti-CD40Lantibodies (mAb FGK45.5 at a concentration of 0.5 μg/ml). Anti-CD40Lantibodies served as a partial substitute for T cell help. Thecombination of sIg and helper T cell like signaling achieved good levelsof B cell activation and proliferation. This could especially bedemonstrated when using LHL-omp at a concentration of 125 ng/ml. 125ng/ml induced no B cell activation on its own, however, when used incombination with the anti-CD40L antibody, which by itself is also of lowpotency, B7-1, B7-2 and FSC upregulation were achieved. Suggesting thatLHL-omp and anti-CD40L antibodies can act synergistically.

Example 30 Utilising OMP to Design a Novel Multimeric Mitogen

Experimental data obtained show that the signal peptide from the outermembrane protein A (ompA) of E. coli induces aggregation of therecombinant proteins LHL.seq and TLHL. The ompA signal peptide (omp) isusually cleaved off once the protein reaches its destination, thebacterial periplasmic space. In the case of LHL, LHL.seq and TLHL,however, the secretion into the periplasm is impaired. All threeproteins remain in the cytoplasm and the omp peptide forms theirN-terminal part. The N-terminal omp peptide induces multimerisation asdemonstrated by the potentiation of their biological activity ascompared to the recombinant protein LHL-omp and TEV-cleaved TLHL.

The observation that omp induces multimerisation allows the design ofsimpler molecules with the same desired biological function as LHL, TLHLand CATAB. For this purpose we propose the following protein design.Above results demonstrate that the proteins described are not secretedinto the periplasmic space. It should therefore be possible to produceproteins that have an omp peptide as their N-terminal part and L or HLas their C-terminal part. As omp allows the formation of multimers, thisshould result in the formation of [ompL]_(n), hereafter called ompL, or[ompHL]_(n) where n is equal or larger than 2.

Example 31 Multimerisation of OMPL and Design of Fv-CATAB

Multimerisation of ompL generates a protein complex that should allowcrosslinking of surface immunoglobulins in a similar fashion to LHL orLHL.seq. OmpL itself, however, is a relatively simple monomeric proteinwhich needs only a single blocking entity. This blocking domain will bethe below described scdsFv resulting the fusion proteinompL-linker-TEV-scdsFv (Fv-catAb). The reverse of this configuration,scdsFv-TEV-linker-Lomp (pFv-catAb) will also be generated, as this mightallow for periplasmic secretion of pFv-catAb. The latter pFv-catAbrequires the functional multimerisation and biological activity of Lomp,a protein with the reverse fusion order of ompL and the omp peptide atits C-terminal. All described recombinant proteins are tested in theexperimental systems outlined above.

Example 32 Redesign of the L Domain Blocking Entity

Two potential problems are associated with the use of the LEN kappalight chain as a blocking domain for L. First, proteins (ie. LHL,LHL.seq and TLHL) are not secreted into the periplasmic space duringexpression in E. coli, which might cause folding problems in the kappaportion. Secondly, there are no direct means of purifying proteins withpotentially correctly folded kappas in the described system, asantibodies against kappa would be bound by LHL.seq.

In order to allow for purification of correctly folded growth factorprecursors, the blocking entity was therefore redesigned. Kappa will bereplaced by a single chain (sc) antibody which is stabilised by aninternal disulphide bridge (disulphide bridge stabilised, ds). ThisscdsFv will be derived from the extensively described plasmacytomaMcPc603 [Freund et al. Biochemistry 33: 3296-3303, 1994] withanti-phosphorylcholine specificity. The phosphorylcholine-bindingability will facilitate the purification of correctly folded recombinantproteins via a phosphorylcholine affinity column.

Example 33 Potential Use of LHL/CATAB Derivatives in Humans

In order to enable production of catalytic antibodies in humans, slightmodifications of the constructs need to be performed. The ‘H’ T cellepitope has to be exchanged for an “universal T cell epitope” which willbe recognised by T cells in the majority of humans in conjunction withtheir more diverse MHC class II molecules.

Example 34 Generation of LgL

The periplasmic secretion of LHL (see PCT/AU97/00194, filed 26 Mar.1997) fusion proteins like TLHL and others demonstrated that the H inLHL was quantitatively cleaved during transport. This made thepurification of full-length products from the periplasmic space or theculture supernatant more difficult. In order to circumvent thisproteolytic cleavage, the H-linker was replaced with a Glycine-Serinelinker. This linker consists of a quadruple repeat of four glycinefollowed by one serine, (GGGGS)×4. In addition the proteins were fusedto a hexa-his-Tag at their C-terminus to allow their purification over anickel-chelate-column (FIG. 1).

Example 35 Structure, Analysis and Purification of LgL

From expression studies with ompL (OHL) the inventors demonstrated thatthe insertion of the H-linker sequence between ompA and L allowedsecretion of L-proteins into the periplasm. In order to direct theexpression of LgL into the periplasmic space, the ompA signal sequenceas well as the H-linker sequence were therefore added to the N-terminusof the protein. This protein was named OHLgL (FIG. 1).

OHLgL was expressed in E. coli strain DH10B by overnight induction with400 μg/l anhydrotetracycline in non-buffered TB-media at roomtemperature. Cells were harvested and incubated in 500 mM sucrose, PBSon ice for 30 min. Cells were pelleted and LgL was purified from thesupernatant containing the periplasmic proteins over a hulgG or anickel-chelate column. LgL containing fractions (FIG. 2) as analysed on20% w/v PHAST-gels were concentrated. LgL was further purified via aSuperose 12 sizing column in PBS. The HPLC Superose 12 sizing profilewas used to determine the concentration of LgL in the final eluateaccording to the absorbance at 280 nm (FIG. 3). LgL containing fractionswere again analysed on 20% w/v PHAST-gels and if necessary pooled for Bcell activation assays (FIG. 4).

Example 36 B Cell Activation Potential of LgL

LgL was tested for its ability to activate B cells as compared tostimulation with anti-IgM and Lomp. Activation status was measured bythe induction of cell surface expression of the activation markers B7-1and B7-2 and by entry of B cells into cell cycle. Levels of expressionof B7-1 and B7-2 were determined by flow cytometry (FACS) withfluorescence-labelled monoclonal antibodies while entry into cell cyclewas monitored by an increase in cell size by Forward Light Scatter(FSC).

FACS were performed as follows. Mesenteric lymph node cells from C3H/HeJmice were centrifuged in Nycodenz (1.091 g/cm³) to remove dead cells andred blood cells (rbc). This was followed by 1 hour adherence on plasticat 37° C. to remove adherent cells such as macrophages. Lymph node cellswere stimulated in triplicate cultures at 3×10⁵/well in flat bottom96-well plates in complete RPMI+10% v/v FCS medium at 37° C. overnight.Upregulation of activation markers on B cells was monitored by gating onB220⁺ Thy⁻ cells to identify B cells. Stimulation with LPS (20 μg/ml)and polyclonal F(ab)₂ anti-IgM antibodies (20 μg/ml) were included ascontrols. LgL was used at 1-10 μg/ml. C3H/HeJ mice were used as sourceof lymphocytes since this particular mouse strain is non-responsive toLPS. The use of this strain in combination with the LPS controleffectively precludes the possibility that B cell stimulation induced byLgL is due to LPS (endotoxin) contamination of the bacterially expressedprotein.

The results of the FACS analysis are as follows. Stimulation of C3H/HeJlymph node cells with LPS did not result in B cell activation whereasstimulation with either anti-IgM antibodies or LgL did as measured byupregulation of B7-1 and B7-2. The characteristic potency of LgL isdemonstrated by the strong induction of B7-1 expression already afterovernight stimulation. Anti-IgM induces B7-1 on day 2-3 afterstimulation (FIG. 5).

Example 37 Generation of ccMTLgL

ccMTLgL was generated by cloning the disulphide linked single chain Fvfrom McPc603 in place of the H sequence in OHLgL. ccM and LgL wereseparated by a glycine-serine linker and the TEV cleavage signal as usedbefore in TLHL. A FLAG-tag was used between the ompA and ccM forpurification purposes. The sequence of the individual protein domainswas therefore as follows: O-FLAG-ccMTLgL-6×his (FIG. 6). The nucleotidesequence and corresponding amino acid sequence for ccMTLgL is set forthin SEQ ID NOs: 15 and 16, respectively.

Example 38 Structure, Analysis and Purification of ccMTLgL

ccMTLgL was expressed in E. coli strain DH10B by overnight inductionwith 400 μg anhydrotetracycline in non-buffered TB-media at roomtemperature. Cells were pelleted and ccMTLgL was purified from theconcentrated supernatant over the Ca⁺⁺ dependent FLAG M1 affinitycolumn. This FLAG M1 affinity column only purifies correctly processedfree FLAG peptide at the N-terminus of a recombinant protein. ccMTLgLcontaining fractions (FIG. 7) as analysed on 20% w/v PHAST-gels wereconcentrated to ≦500 μl in 10.000 MW cut off spin concentrator. ccMTLgLwas further purified via a Superose 12 sizing column in PBS. The HPLCSuperose 12 sizing profile was used to determine the concentration ofccMTLgL in the final eluate according to the absorbance at 280 nm (FIG.8). ccMTLgL containing fractions were again analysed on 20% w/vPHAST-gels and if necessary pooled for B cell activation assays (FIG.9). The correct formation of the inter-domain disulphide bond was shownby running ccMTLgL on 20% w/v PHAST-gel under reducing and non-reducingcondition before and after cleavage with TEV (FIG. 10).

Example 39 TEV Catalysis Induced B Cell Activation by ccMTLgL

25 μg of ccMTLgL in 140 μl of PBS were incubated with 50 Units TEVprotease at 4° C. overnight. Complete cleavage into ccMT and LgL wasverified on a 20% w/v PHAST-gel (FIG. 10).

Mesenteric LN cells (prepared as above) were stimulated overnight withcontrols (anti-IgM, LPS, LOMP, LgL and 2.5 U TEV protease alone; allwith and without hulgG) as well as 10 μg/ml ccMTLgL and 10 μg/ml ccMTLgLcleaved with TEV.

Results are shown in FIG. 11. ccMTLgL by itself gives no B cellstimulation whereas ccMTLgL cleaved with TEV shows B cell stimulationwith upregulation of B7-1.

These results were reproduced three times. The same results were alsoobtained when 2.5 U TEV protease were added in situ to the o/n B cellcultures (FIG. 12). Demonstrating that the in situ cleavage of ccMTLgLhas the desired effect of liberating a B cell mitogen. This mimics theaction of a catalytic antibody expressed by a B cell.

Example 40 Utilising OMP to Design a Novel Multimeric Mitogen

ompL (FIG. 13) is secreted into the periplasmic space. The ompA signalpeptide is, therefore, processed and cleaved off. ompL can be purifiedon a hulgG column. ompL fractions from hulgG column are concentratedover a Millipore concentrator and are further purified over aSuperose-12 HPLC sizing column. ompL does not multimerise and,therefore, runs as a monomeric protein at approximately 10 kD.

Lomp is the reverse of ompL, carrying a modified ompA signal peptide atthe C-terminus of LH. Lomp is expressed intracellularly and purified viahulgG and Superose-12 as described for ompL. ompL multimerises aspredicted and elutes from the HPLC column in the void volume at ≧670 kD.

ompL and Lomp were tested for their ability to activate B cells. Asmeasured by the induction of cell surface expression of activationmarkers and by entry into cell cycle. The method is as described above.

FACS analysis showed that this two day stimulation of lymph node cellswith LPS did not result in B cell activation whereas stimulation witheither anti-IgM antibodies or Lomp did as measured by an increased FSCand up regulation of B7-2. The characteristic potency of Lomp isdemonstrated by the strong induction of B7-1 expression afterincubation.

Lomp activity was blocked by the addition of 500 μg/ml soluble hulgGinto the culture.

ompL has no activity in FACS or proliferation assays.

Example 41 Re-Design of the L Domain Blocking Entity

A single chain Fv of McPc603 [scMcPc603] is expressed into theperiplasmic space of E. coli DH10B. scMcPc603 can be purified on aL-column (FIG. 15). scMcPc603 is properly folded because it binds to theL domain. scMcPc603 can be utilised as a blocking entity for L in acatab construct. In one example, Fv-catAb is used (FIG. 14).

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

1-16. (canceled)
 17. A catalytic antibody generated according to themethod of claim 27 or
 28. 18. A nucleic acid molecule according to anyone of claims 20-22 further comprising a nucleotide sequence encoding amolecular adjuvant.
 19. A nucleic acid molecule according to claim 18wherein the molecular adjuvant is selected from C3d, CTLA4 and CD40L.20. An isolated nucleic acid molecule comprising a sequence ofnucleotides encoding a recombinant growth factor precursor, wherein saidrecombinant growth factor precursor comprises a recombinant polypeptidechain or a molecule having modular peptide components or a syntheticequivalent thereof, wherein said polypeptide chain or modular peptidemolecule comprises at least one B cell surface molecule binding portion,at least one T cell surface molecule binding portion capable ofproviding T cell dependent help to a B cell, an antigen cleavable by acatalytic antibody and a peptide portion comprising domains from both avariable heavy chain and a variable light chain of an immunoglobulin andwherein said variable heavy chain and variable light chain domains inthe growth factor precursor, associate together by intra- and/orinter-domain bonding and substantially prevent the at least one B cellsurface molecule binding portion from interacting with a B cell surfacemolecule such that upon cleavage of said antigen by a catalyticantibody, the peptide comprising said variable heavy chain and variablelight chain domain permits the at least one B cell surface moleculebinding portion to interact with a B cell surface molecule.
 21. Anisolated nucleic acid molecule comprising a sequence of nucleotidesencoding a recombinant growth factor precursor, wherein said recombinantgrowth factor precursor comprises a recombinant polypeptide chain or amolecule having modular peptide components or a synthetic equivalentthereof, wherein said polypeptide chain or modular peptide moleculecomprises at least one B cell surface molecule binding portion, at leastone T cell surface molecule binding portion capable of providing T celldependent help to a B cell, an antigen cleavable by a catalytic antibodyand a peptide portion comprising domains from both a variable heavychain and a variable light chain of an immunoglobulin and wherein saidvariable heavy chain and variable light chain domains in the growthfactor precursor associate together by intra- and/or inter-domainbonding and substantially prevent the at least one B cell surfacemolecule binding portion from interacting with a B cell surface moleculesuch that upon cleavage of said antigen by a catalytic antibody, thepeptide comprising said variable heavy chain and variable light chaindomain permits the at least one B cell surface molecule binding portionto interact with a B cell surface molecule wherein if said growth factorprecursor comprises a single B cell surface molecule binding portion,then the growth factor precursor further comprises a multimerizinginducing element.
 22. An isolated nucleic acid molecule comprising asequence of nucleotides encoding a recombinant growth factor precursor,wherein said recombinant growth factor precursor comprises thestructure:I′AX₁[X₂]_(d)[X₃]_(a)[A]_(r)I″ wherein: X₁ and X₃ are B cell surfacemolecule binding portions; a is 0 or 1 or >1; I′ and I″ are either bothpresent or only one is present and they may be the same or different andeach is a blocking reagent for X₁ and/or X₃ and comprise domains fromboth a variable heavy chain and a variable light chain of animmunoglobulin, and wherein said variable heavy chain and variable lightchain domains associate together by intra- and/or inter-domain bondingand substantially prevent at least one of X₁ or X₃ from interacting witha B cell surface molecule; A is the target antigen for which a catalyticantibody is sought; X₂ is an entity providing T cell dependent help to aB cell; d is 0, 1 or >1; r is 0, 1 or >1, wherein a catalytic antibodyon the surface of said B cell is capable of cleaving all or part of Afrom said recombinant or synthetic molecule resulting in the molecule[A′]X₁X₂[X₃]_(a)[A′] wherein A′ is optionally present and is a portionof A after cleavage with the catalytic antibody wherein said resultingmolecule is capable of interacting with said B cell surface moleculethrough at least one of X₁ or X₃ and inducing B cell mitogenesis of theB cell to which X₁ or X₃ bind.
 23. A composition comprising arecombinant or synthetic growth factor precursor and at least one of apharmaceutical carrier and a diluent, wherein said recombinant orsynthetic growth factor precursor comprises a recombinant polypeptidechain or a molecule having modular peptide components or a syntheticequivalent thereof, wherein said polypeptide chain or modular peptidemolecule comprises at least one B cell surface molecule binding portion,at least one T cell surface molecule binding portion capable ofproviding T cell dependent help to a B cell, an antigen cleavable by acatalytic antibody and a peptide portion comprising domains from both avariable heavy chain and a variable light chain of an immunoglobulin andwherein said variable heavy chain and variable light chain domains inthe growth factor precursor, associate together by intra- and/orinter-domain bonding and substantially prevent the at least one B cellsurface molecule binding portion from interacting with a B cell surfacemolecule such that upon cleavage of said antigen by a catalyticantibody, the peptide comprising said variable heavy chain and variablelight chain domain permits the at least one B cell surface moleculebinding portion to interact with a B cell surface molecule.
 24. Acomposition comprising a recombinant or synthetic growth factorprecursor and at least one of a pharmaceutical carrier and a diluent,wherein said recombinant or synthetic growth factor precursor comprisesa recombinant polypeptide chain or a molecule having modular peptidecomponents or a synthetic equivalent thereof, wherein said polypeptidechain or modular peptide molecule comprises at least one B cell surfacemolecule binding portion, at least one T cell surface molecule bindingportion capable of providing T cell dependent help to a B cell, anantigen cleavable by a catalytic antibody and a peptide portioncomprising domains from both a variable heavy chain and a variable lightchain of an immunoglobulin and wherein said variable heavy chain andvariable light chain domains in the growth factor precursor associatetogether by intra- and/or inter-domain bonding and substantially preventthe at least one B cell surface molecule binding portion frominteracting with a B cell surface molecule such that upon cleavage ofsaid antigen by a catalytic antibody, the peptide comprising saidvariable heavy chain and variable light chain domain permits the atleast one B cell surface molecule binding portion to interact with a Bcell surface molecule wherein if said growth factor precursor comprisesa single B cell surface molecule binding portion, then the growth factorprecursor further comprises a multimerizing inducing element.
 25. Acomposition comprising a catalytic antibody generated to a growth factorprecursor, wherein said growth factor precursor comprises a recombinantpolypeptide chain or a molecule having modular peptide components or asynthetic equivalent thereof, wherein said polypeptide chain or modularpeptide molecule comprises at least one B cell surface molecule bindingportion, at least one T cell surface molecule binding portion capable ofproviding T cell dependent help to a B cell, an antigen cleavable by acatalytic antibody and a peptide portion comprising domains from both avariable heavy chain and a variable light chain of an immunoglobulin andwherein said variable heavy chain and variable light chain domains inthe growth factor precursor, associate together by intra- and/orinter-domain bonding and substantially prevent the at least one B cellsurface molecule binding portion from interacting with a B cell surfacemolecule such that upon cleavage of said antigen by a catalyticantibody, the peptide comprising said variable heavy chain and variablelight chain domain permits the at least one B cell surface moleculebinding portion to interact with a B cell surface molecule.
 26. Acomposition comprising a catalytic antibody generated to a growth factorprecursor, wherein said growth factor precursor comprises a recombinantpolypeptide chain or a molecule having modular peptide components or asynthetic equivalent thereof, wherein said polypeptide chain or modularpeptide molecule comprises at least one B cell surface molecule bindingportion, at least one T cell surface molecule binding portion capable ofproviding T cell dependent help to a B cell, an antigen cleavable by acatalytic antibody and a peptide portion comprising domains from both avariable heavy chain and a variable light chain of an immunoglobulin andwherein said variable heavy chain and variable light chain domains inthe growth factor precursor associate together by intra- and/orinter-domain bonding and substantially prevent the at least one B cellsurface molecule binding portion from interacting with a B cell surfacemolecule such that upon cleavage of said antigen by a catalyticantibody, the peptide comprising said variable heavy chain and variablelight chain domain permits the at least one B cell surface moleculebinding portion to interact with a B cell surface molecule wherein ifsaid growth factor precursor comprises a single B cell surface moleculebinding portion, then the growth factor precursor further comprises amultimerizing inducing element.
 27. A method of inducing B cellmitogenesis to generate a catalytic antibody to a specific antigencomprising administering to an animal a growth factor precursor, whereinsaid growth factor precursor comprises a recombinant polypeptide chainor a molecule having modular peptide components or a syntheticequivalent thereof, wherein said polypeptide chain or modular peptidemolecule comprises at least one B cell surface molecule binding portion,at least one T cell surface molecule binding portion capable ofproviding T cell dependent help to a B cell, said antigen cleavable bysaid catalytic antibody, and a peptide portion comprising domains fromboth a variable heavy chain and a variable light chain of animmunoglobulin wherein said variable heavy chain and variable lightchain domains associate together by intra- and/or inter-domain bondingand substantially prevent the at least one B cell surface moleculebinding portion from interacting with a B cell surface molecule suchthat upon cleavage of said antigen by said catalytic antibody, thepeptide comprising said variable heavy chain and variable light chaindomain permits the at least one B cell surface molecule binding portionto interact with a B cell surface molecule to induce B cell mitogenesisthereby generating said catalytic antibody to said antigen.
 28. A methodof inducing B cell mitogenesis to generate a catalytic antibody to aspecific antigen comprising administering to an animal a growth factorprecursor, wherein said growth factor precursor comprises a recombinantpolypeptide chain or a molecule having modular peptide components or asynthetic equivalent thereof, wherein said polypeptide chain or modularpeptide molecule comprises at least one B cell surface molecule bindingportion, at least one T cell surface molecule binding portion capable ofproviding T cell dependent help to a B cell, said antigen cleavable bysaid catalytic antibody and a peptide portion comprising domains fromboth a variable heavy chain and a variable light chain of animmunoglobulin and wherein said variable heavy chain and variable lightchain domains in the growth factor precursor associate together byintra- and/or inter-domain bonding and substantially prevent the atleast one B cell surface molecule binding portion from interacting witha B cell surface molecule such that upon cleavage of said antigen bysaid catalytic antibody, the peptide comprising said variable heavychain and variable light chain domain permits the at least one B cellsurface molecule binding portion to interact with a B cell surfacemolecule to induce B cell mitogenesis thereby generating said catalyticantibody to said antigen, and wherein if said growth factor precursorcomprises a single B cell surface molecule binding portion, then thegrowth factor precursor further comprises a multimerizing inducingelement.